Formulation of microorganisms for use in bait gels for rodent control

ABSTRACT

The present invention relates to compositions containing different stages of pathogenic protozoans in a gel and to their use for controlling rodents.

[0001] The present invention relates to compositions containingdifferent stages of pathogenic protozoans in a gel and to their use forcontrolling rodents.

[0002] It is known that protozoa from the group of the Sarcosporidia(phylum Protozoa, class Sporozoa, subclass Coccidia, order Eucoccidiida,suborder Eimeriina) are pathogenic to rodents (Genera Rattus, Bandicota,Arvicanthis, Nosokia, Mus) and that a sufficient dose leads to theanimals' death (Rzepczyk 1974, Intern. J. Parasitol. 4, 447-449; Brehm,Frank 1980, Z. Parasitenkd. 62, 15-30). A parasite from among this groupwhich is especially pathogenic to several rat species is Sarcocystissingaporensis (Zamann et al. 1975, Z. Parasitenkd. 47, 169-185).

[0003] The definite hosts of the parasites are predators or omnivores;for S. singaporensis, the definite hosts are snakes, for example thereticulated python. The parasite forms sporocysts in the gut of thedefinite host. These persistent stages are excreted together with thefaeces. If an intermediate host, in the case of S. singaporensis forexample a rat, is infected orally with these sporocysts, sporozoiteshatch in the rat's intestines and penetrate the intestinal wall. Via theblood circulation, these stages reach other organs, where they canmultiply by division (Schizogony).

[0004] Two schizogonies have been detected, the first one around day 6post-infection and the second around day 14-16 post-infection. Theschizonts (merozoites) are found in particularly high numbers in thelungs, where they multiply in the endothelial cells.

[0005] If severe infection results in mass multiplication of themerozoites in the lung, the rat falls ill during the second schizogonyof the parasite around day 16 post-infection and, in most cases, dieswithin a few hours.

[0006] It is possible to administer the sporocysts of these pathogens torodents so that the latter die from the infection. This principle can beexploited for controlling rodents (Wood 1985, J. Plant Protect. Trop. 2,67-69). Rats have already been controlled successfully with Sarcocystissingaporensis (Jakel et al. 1996, J. Parasitol. 82, 280-287).

[0007] As a rule, infections of Rattus rattus and norvegicus with over20,000 Sporocysts (depending on the strain) are lethal. Up to 14 dayspost-infection the animals show no symptoms. Then, they weaken rapidlyand die of the infection around day 16 post-infection.

[0008] Sublethally infected rats show virtually no symptoms of disease,even after 14 days post-infection, and survive the infection withoutloss of vitality. Only a few days after the infection, these animalshave established immunity to the pathogen. These animals are nowinsensitive to any further control with a Sarcocystis singaporensisproduct.

[0009] The sporozoites in aqueous suspension have a shelf life of up totwo years without substantial loss of pathogenicity. This suspension canbe administered to rats by gavage and, with appropriate dosing, has alethal effect. However, the sporocysts do not survive in the dry state.

[0010] To control rats, baits must be used which are eaten by theanimals. Such baits with S. singaporensis sporocysts remain pathogenicas long as they are moist. Once dried out, the sporocysts very rapidlylose their pathogenicity. This is why conventional cereal-based baitscannot be used for the effective administration of these parasites. Inthe applicant's own studies it was impossible to retain the infectivityof S. singaporensis sporocysts in cereal-based baits. One day aftermixing, these baits had become ineffective.

[0011] Baits with sporocysts must be film-packaged in the moist state.The disadvantage of these moist products is the low shelf life sincecereal would start to go mouldy, or would germinate, within a few days.One alternative is to dose the liquid suspension in cavities in thebait. For example, lumps of pasty bait whose interior contained a lethaldose of the suspension in a cavity were used successfully. However,these baits too must be used within a few days after preparation sincewater escapes or since the pasty matrix absorbs water and swells orferments, rendering these baits useless.

[0012] The only possibility as yet for the practical use of protozoasuch as S. singaporensis for rodent control is to prepare the baitsimmediately prior to use from a sporocyst-containing aqueous suspensionand a bait matrix, for example cereals. The problem here is to withdrawa calculated amount of sporocysts since the latter settle very rapidlyin the suspension and concentrate at the bottom of the vessel. This iswhy baits prepared in this manner contain an indeterminate amount ofsporocysts which is not distributed homogeneously. These baits dryrapidly, frequently even during mixing. As a consequence, the sporocystsdie and the bait stops being effective after as little as a few hours.

[0013] It has now been found, surprisingly, that Sarcocystis sporocystsretain their infectivity for a long time in specific gels, even thoughit is known from earlier studies that they are no longer infectious oncereals, in pastes and gels such as bentonites after as little as a fewdays. The fact that they survive over months in the gels describedherein was very surprising and unexpected. It is known from theliterature that even highly robust sarcosporidia from temperateclimates, such as S. gigantea, are very sensitive to changes in the pHand chemicals (McKenna et al., Veterinary Parasitology 1992, 45 1-16).It is therefore particularly surprising that even the pH can be variedand in addition preservatives can be added without substantiallyreducing the parasite's chances of survival.

[0014] The present invention therefore makes it possible to preservesporocysts in gels for the later use in the control of rodents. Theyremain distributed homogeneously in this matrix. This makes possible thewithdrawal of defined quantities of sporocysts. A defined quantity ofthis gel can be mixed with the bait matrix as long as a homogeneousstate has been achieved since the gel is only adsorbed very slowly bythe matrix. Since the loss of water, by evaporation, from the gels usedis only very slow, the bait remains moist, and thus infectious, evenwhen used in a dry atmosphere.

[0015] In this manner, it is possible to store sporocysts over monthswithout suffering gel separation or a considerably reduced infectivityof the pathogens. The appended example, in which a Carbopol gel is used,confirms that such mixtures can be used over several months.

[0016] The present invention claims inter alia the principle ofpreserving stages of single-celled parasites in gels. These gels areused for controlling rodents, either alone or admixed to baits.

[0017] Suitable pathogenic organisms are, in principle, all protozoafrom the class Sporozoa. Especially suitable are species from thesubclass Coccidia. Very specially suitable are representatives of thesuborder Eimeriina. Examples which may be mentioned, but not bylimitation, are the species of the genus Sarcocystis, such as S.singaporensis.

[0018] The composition according to the invention can be incorporatedinto all formulas conventionally used for the production of feed baitsfor rodents, for example into cereal-based baits, into pastes, gels andwax blocks and into extruded mixtures of cereals and formulationauxiliaries. It is furthermore possible to offer the pathogens inwater-based drink baits. The baits for the purposes of the invention areused like conventional baits for controlling harmful rodents. They canbe placed in enclosed spaces and in the open, with 5 g to 500 g per baitstation, depending on the target species and the bait station density.They can also be placed in the burrow entrances of the rodents to becontrolled.

[0019] Dosing of the pathogens in the gel can be set as desired. Dosagesof from 1000 to 10,000,000 sporocysts/ml are to be preferred. 500,000 to2,000,000 sporocysts/ml are especially preferred.

[0020] The dosage of the pathogens in the bait should amount to 1000 to200,000 sporocysts per gram of bait. Concentrations of from 2000 to100,000 sporocysts per gram are preferred, concentrations of10,000-30,000 are especially preferred and 20,000 sporocysts per gramare particularly preferred. Higher dosages are also possible, but, as arule, not required. It is possible to employ sporocysts of only onepathogen species or else mixtures of sporocysts of different pathogens.

[0021] Harmful and other rodents to be controlled are, for the purposesof the invention, rodents from the order Rodentia. The genera Rattus,Mus, Bandicota, Nosokia and Microtus are particularly emphasized. Therepresentatives of the genera Rattus and Bandicota, e.g. R. rattus, R.norvegicus, B. benigalensis, B. indica, are especially to be emphasized.

[0022] Gels for the purposes of the invention are water-based andtreated with suitable thickeners. Suitable thickeners are macromoleculessuch as cellulose derivatives, for example hydroxypropylcellulose,hydroxyethylcellulose, methylcellulose, carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylmethylcellulose,hydroxyethylpropylcellulose or xanthans, alginates, polyvinyl alcohols,polyvinylpyrrolidone, polyacrylic acids and their derivatives, orinorganic gellants such as highly-dispersed silica (see, for example,Rudolf Voigt, Pharmazeutische Technologie [Pharmaceutical Technology],pages 362-385, Ulstein Mosby). Particularly suitable are cellulosederivatives and polyacrylic acids.

[0023] The water content of the gels can be varied within wide ranges,for example between 30 and 98% by weight, in particular between 50 and90% by weight and very especially between 70 and 80% by weight.

[0024] The pH of the gels can also be varied within wide ranges, forexample between pH 2 and 10, in particular between pH 4 and 9,especially preferably between pH 6 and 8.

[0025] The gels can be treated with preservatives such as parabens,benzoates, sorbic acid, citrates or parabens, humectants such asglycerol or propylene glycol, antioxidants such as butylhydroxytolueneor butylhydroxyanisole, tocopherol, ascorbic acid, flavourings or otherformulation auxiliaries.

[0026] In the compositions according to the invention, the sporocysts ofthe pathogenic protozoa can also be combined with further, including,for example, chemical, active compounds for rodent control. Thecoumarins may be mentioned here by way of example, but not bylimitation.

[0027] The compositions according to the invention are prepared bymixing the components in the relevant proportions. Mixing is performedmechanically, for example in stirred vessels or other apparatuses whichare suitable for this purpose.

[0028] The examples which follow are intended to illustrate the presentinvention, but without imposing any limitation.

EXAMPLE

[0029] Method

[0030] The used sporocysts of strain S5 (available at HohenheimUniversity, Parasitology Department) originate from a Passage inThailand dated Aug. 17, 1998. A Neubauer haematocytometer was used forcounting. The gels were prepared with 1% Carbopol 974 P (Carbopols:weakly crosslinked polyacrylic acids) in water (342 ppm). 2N NaOH wasadded by titration to adjust the pH. The preservatives used were 0.02%of Solbrol P and 0.14% of Solbrol M (Solbrols: para-hydroxybenzoic acidesters (methyl, ethyl, butyl)). One sample contained 20% of glycerol.50,000 sporocysts per ml were added to all gels and the gels were mixedthoroughly in order to achieve homogeneous distribution. The gels werekept in the refrigerator at approx. 6° C. and administered at therespective experimental days in accordance with the experimentalschedule.

[0031] All the experimental animals used were wild R. norvegicus. 14hours prior to being offered the sporocyst bait, the animals werestarved and had ad libitum access to water. The bait was based oncrushed wheat. Each rat was given 5 g of crushed wheat treated with 1 mlof gel. The rats were again offered standard feed ad libitum only afterthey had eaten the Sarcocystis bait. Two rats which were observed up to21 days after dosing were used for each dosage. The parametersdetermined were the mortality as a function of the gel formulation andthe storage period. Rats which showed severe symptoms within the periodduring which an effect of the pathogen was expected (days 13-17) whichwould make survival under natural conditions unlikely were destroyed.

[0032] Results TABLE 1 Mortality after the uptake of bait with 50,000sporocysts (* with 100,000) in various gels on crushed wheat as afunction of gel storage time. Gel Mortality in (%) after a week'sstorage time pH Solbrol Glycerol 0 2 4 6 8 10 12 14 19 24 6.17 + − 100100 100 50 0 7.04 + + 100 50 0 0 0 7.05 − − 100 100 100 0 50* 8.19 + −100 100 100 0 0

1. Composition containing sporocysts of pathogenic protozoa in anaqueous gel.
 2. Composition according to claim 1, characterized in thatthe aqueous gel is based on water and a thickener from the groupconsisting of cellulose derivatives, xanthans, alginates, polyvinylalcohols, polyvinylpyrrolidone, polyacrylic acids and inorganicgellants.
 3. Compositions according to claims 1 and 2, characterized inthat they contain spoiocysts of pathogenic protozoa at a concentrationof 1000 to 10,000,000 per ml.
 4. Compositions according to claims 1 and2, characterized in that they contain between 30% and 98% of water. 5.Composition for controlling rodents, characterized in that it containsthe compositions according to claims 1 and 2 in addition to thecustomary bait materials.
 6. Use of compositions according to claims 1and 2 for the preparation of baits for controlling rodents,characterized in that the compositions are mixed with customary baitmaterials.
 7. Use of compositions according to claims 1, 2 and 5 forcontrolling rodents, characterized in that the compositions are placedin the rodents' environment.